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Assessment of laboratory tests efficiency in case of exocrine pancreas function determination
Šubčíková, Lenka ; Martínková, Markéta (advisor) ; Milichovský, Jan (referee)
Chronic pancreatitis is complex disease with complicated diagnosis. Nowadays there are the diagnosis and classification of chronic pancreatitis primarily based on imaging methods. In this study the results of two indirect tests of pancreatic exocrine function with different arrangement were compared. The pancreatic fecal elastase 1 was determinate by immunoassay with monoclonal antibody, as a simple screening test. The dynamics and kinetics of pancreatic exocrine secretion was observed by a breath test with 13 C-labeled substrate. The group of four volunteers was monitored for six months and analyzed each month. The mutual variability of these tests and their correlation was studied. The patients' (suspected suffering by chronic pancreatitis) anonymous data for these tests created by the Institute of laboratory biochemistry and laboratory diagnostics between 1999-2012 were statistical processed. We found, that the indirect tests of pancreatic exocrine function and determination of pancreatic enzymes does not correlate. Both of these tests have the specific diagnostic value mainly for diagnosis of pancreatic exocrine insufficiency. 1
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Heterologous expression and isolation of human cytochromes P450 1A1/2
Milichovský, Jan ; Martínek, Václav (advisor) ; Hodek, Petr (referee)
Cytochromes P450 form a large family of hemoproteins. Some of them are responsible for the metabolism of endogenous substrates, but their major role is in detoxification of exogenous substrates (xenobiotics), some of them are activated to reactive species forming covalent adducts with DNA and increasing intracellular oxidative stress. Cytochrome P450 are considered by very promiscuous in terms of their substrate specificity, thus one enzyme can typically oxidize many substrates. Cytochrome P450 1A1 prefers a planar aromatic compounds (e.g. polycyclic aromatic hydrocarbons, azo dyes, etc.). Cytochrome P450 1A2 elicits similar substrate specificity, but prefers slightly basic aromatic derivatives, for example caffeine. This work focuses on (i) the preparation of expression vectors containing genes encoding human cytochromes P450 1A1 and 1A2, (ii) their consequent expression in heterologous system followed by (iii) isolation of corresponding proteins. The genes coding both proteins were modified and transferred from older vectors to the more efficient to expression plasmids pET-22b. However, the new constructs did not produce stable native proteins. The modified genes were therefore transferred to the original expression plasmids pCW. The problem with the incorporation of native human form of...
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Preparation of expression vectors for receptor NKp30 and its ligands B7-H6 and BAG-6
Pažický, Samuel ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
NK cells, the cells of non-adaptive imune system, are able to recognise viraly infected or oncogenous cells by many inhibiting and activating receptors that are expressed on their surface and eliminate them consequently. For NKp30, activating receptor of NK cells included in NCR (Natural Killer Cell Receptors) family, lately there were identified several ligands, including membrane protein B7-H6 expressed on oncogenous cells surface and BAG- 6, cell core protein with wide spectrum of functions. Aim of this thesis was preparation of expression vectors coding for receptor NKp30 and his ligands B7-H6 and BAG-6, enabling expression of these proteins in HEK293 cell line. Keywords: NK cell, plasmid, receptor, NKp30, B7-H6, BAG-6
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Molecular mechanisms of apoptosis induction in cancer cells.
Fenclová, Tereza ; Novák, Petr (advisor) ; Milichovský, Jan (referee)
Cancer diseases are now the third leading cause of death (20% of all deaths). It is therefore important to find new ways of getting tumor cells effectively and specifically disposed of and a promising path is targeted therapy. One of the most frequently deregulated protooncogenes in cancer is C-MYC, which makes it suitable as an effective target for treatment. However, the development of such targeted active ingredients is very expensive, so in this thesis we investigate natural substances that have been used for the treatment of cancer in ancient China. We examined the substances shikonin, cnicin and artemisinin. The results show that shikonin induces apoptosis of tumor cells by reducing the expression of C-MYC and activating tumor suppressor kinase MST1. Cnicin reduces the expression of C-MYC as well, but activates MST1 only weakly. Artemisinin, on the other hand, increases expression of C-MYC and doesn't activate MST1, thus operates on inducing apoptosis of tumor cells by a completely different mechanism. (In Czech)
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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan
Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...
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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan
Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...
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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan ; Martínek, Václav (advisor) ; Befekadu, Asfaw (referee) ; Souček, Pavel (referee)
Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5 can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...
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Preparation of lentiviral expression vector with reporter gene
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
Besides recombinant protein expression in prokaryotic cell lines (E. coli), systems, that could quickly, reliably and stably produce recombinant proteins in human cell lines, come to the fore. These cell lines assure proper tertiary structure and post-translational modification of the desired products. One of the ways to achieve production of recombinant proteins in human cell lines is the use of lentiviral vectors. This thesis describes the preparation of the lentiviral vector (plasmid) Daedalus, which contains a construct for recombinant expression of secreted alkaline phosphatase. For the preparation of the desired plasmid methods based on insertion of the secreted alkaline phosphatase gene using the restriction endonucleases and methods based on amplification by polymerase chain reaction (restriction-free cloning, transfer polymerase chain reaction and Gibson assembly) were used.
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Preparation of expression vectors for receptor NKp30 and its ligands B7-H6 and BAG-6
Pažický, Samuel ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
NK cells, the cells of non-adaptive imune system, are able to recognise viraly infected or oncogenous cells by many inhibiting and activating receptors that are expressed on their surface and eliminate them consequently. For NKp30, activating receptor of NK cells included in NCR (Natural Killer Cell Receptors) family, lately there were identified several ligands, including membrane protein B7-H6 expressed on oncogenous cells surface and BAG- 6, cell core protein with wide spectrum of functions. Aim of this thesis was preparation of expression vectors coding for receptor NKp30 and his ligands B7-H6 and BAG-6, enabling expression of these proteins in HEK293 cell line. Keywords: NK cell, plasmid, receptor, NKp30, B7-H6, BAG-6
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